Enveloped, negative-sense, single stranded RNA viruses are uniquely organized and expressed. The genomic RNA of negative-sense, single stranded viruses serves two template functions in the context of a nucleocapsid: as a template for the synthesis of messenger RNAs (mRNAs) and as a template for the synthesis of the antigenome (+) strand. Negative-sense, single stranded RNA viruses encode and package their own RNA-dependent RNA Polymerase. Messenger RNAs are only synthesized once the virus has entered the cytoplasm of the infected cell. Viral replication occurs after synthesis of the mRNAs and requires the continuous synthesis of viral proteins. The newly synthesized antigenome (+) strand serves as the template for generating further copies of the (−) strand genomic RNA.
The etiological agent of mumps was first shown reproducibly to be a virus by Johnson and Goodpasture in 1935 (Johnson and Goodpasture, 1935). Since then, propagation in tissue culture has facilitated virus classification and studies on the biological properties of mumps virus (MUV). Originally classified with influenza viruses in the Myxovirus family, mumps virus has since been re-assigned to the Paramyxoviridae family, subfamily Paramyxovirinae, genus Rubulavirus, based on nucleocapsid morphology, genome organization and biological properties of the proteins. Other examples of the Rubulavirus genus include simian virus 5 (SV5), human parainfluenza virus type 2 and type 4 and Newcastle disease virus (Lamb and Kolakofsky, 1996). Like all viruses of the Paramyxoviridae, mumps virus is pleomorphic in shape, comprising a host cell derived lipid membrane surrounding a ribonucleoprotein core; this nucleocapsid core forms a helical structure composed of a 15,384 nucleotide nonsegmented negative sense RNA genome closely associated with virus nucleocapsid protein (NP). The genetic organization of the MUV genome has been determined to be 3′-NP-P-M-F-SH-HN-L-5′ (Elango et al., 1998). Each gene encodes a single protein except for the P cistron, from which three unique mRNAs are transcribed; one is a faithful copy of the P gene, encoding the V protein, the two other mRNAs contain two and four non-templated G residues inserted during transcription by a RNA editing mechanism, and encode the P and I proteins respectively (Paterson and Lamb, 1990). The P and L proteins in association with nucleocapsid form the functional RNA polymerase complex of mumps virus. The F and HN proteins are integral membrane proteins which project from the surface of the virion, and are involved in virus attachment and entry of cells. The small hydrophobic protein (SH) and matrix (M) protein are also membrane associated (Takeuchi et al, 1996 and Lamb and Kolakofsky, 1996); the role of the V and I proteins in virus growth is not yet clear.
The replicative cycle of mumps virus initiates upon fusion of virus envelope with host cell plasma membrane and subsequent release of virus nucleocapsid into the cell cytoplasm. Primary transcription then ensues, resulting in the production of all virus proteins; a switch to replication of the virus genome occurs later, followed by assembly of virus components to form new virus particles which bud from the host cell plasma membrane. Only the intact nucleocapsid structure can act as the template for RNA transcription, replication and subsequent virus amplification; therein lies the difficulty in genetic manipulation of MUV and other negative strand RNA viruses. Unlike the positive strand RNA viruses where naked genomic RNA is infectious and infectious virus can be recovered from a cDNA copy of the genome in the absence of additional viral factors (Taniguchi et al., 1978; Racaniello and Baltimore, 1981), the naked genome of negative strand RNA viruses is not infectious and rescue of virus from cDNA requires intracellular co-expression of viral NP, P and L proteins, along with a full length positive sense, or negative sense, genome RNA transcript, all under control of the bacteriophage T7 RNA polymerase promoter (Schnell et al., 1994; Lawson et al. 1995; Whelan et al., 1995; Radecke et al., 1995; Collins et al., 1995; Hoffman and Banerjee, 1997; Durbin et al., 1997; He et al., 1997; Baron and Barrett, 1997; Jin et al., 1998; Buchholz et al., 1999; Peeters et al., 1999). In all of the reported systems T7 RNA polymerase has been supplied either by a co-infecting recombinant vaccinia virus (Fuerst et al., 1986; Wyatt et al., 1995), or by endogenous expression of T7 RNA polymerase in a transformed cell line (Radecke et al., 1995).
The polymerase complex actuates and achieves transcription and replication by engaging the cis-acting signals at the 3′ end of the genome, in particular, the promoter region. Viral genes are then transcribed from the genome template unidirectionally from its 3′ to its 5′ end. There is generally less mRNA made from the downstream genes (e.g., the polymerase gene (L)) relative to their upstream neighbors (i.e., the nucleoprotein gene (NP)). Therefore, there is always a gradient of mRNA abundance according to the position of the genes relative to the 3′-end of the genome.
Molecular genetic analysis of such nonsegmented RNA viruses has proved difficult until recently because naked genomic RNA or RNA produced intracellularly from a transfected plasmid is not infectious (Boyer and Haenni, 1994). These methods are referred to herein as “rescue”. There are publications on methods of manipulating cDNA rescue methods that permit isolation of some recombinant nonsegmented, negative-strand RNA viruses (Schnell et al., 1994). The techniques for rescue of these different negative-strand viruses follows a common theme; however, each virus has distinguishing requisite components for successful rescue (Baron and Barrett, 1997; Collins et al., 1995; Garcin et al., 1995; Hoffman and Banerjee, 1997; Lawson et al., 1995; Radecke et al., 1995; Schneider et al., 1997; He et al, 1997; Schnell et al., 1994; Whelan et al., 1995). After transfection of a genomic cDNA plasmid, an exact copy of genome RNA is produced by the combined action of phage T7 RNA polymerase and a vector-encoded ribozyme sequence that cleaves the RNA to form the 3′ termini. This RNA is packaged and replicated by viral proteins initially supplied by co-transfected expression plasmids. In the case of the mumps virus, a method of rescue has yet to be established and accordingly, there is a need to devise a method of mumps rescue. Devising a method of rescue for mumps virus is complicated by the absence of extensive studies on the biology of mumps virus, as compared with studies on other RNA viruses. Also, mumps virus does not grow efficiently in tissue culture systems. Furthermore, the sequence for the termini of the mumps virus genome has not previously been characterized in sufficient detail for conducting rescue.
For successful rescue of mumps virus from cDNA to be achieved, numerous molecular events must occur after transfection, including: 1) accurate, full-length synthesis of genome or antigenome RNA by T7 RNA polymerase and 3′ end processing by the ribozyme sequence; 2) synthesis of viral NP, P, and L proteins at levels appropriate to initiate replication; 3) the de novo packaging of genomic RNA into transcriptionally-active and replication-competent nucleocapsid structures; and 4) expression of viral genes from newly-formed nucleocapsids at levels sufficient for replication to progress.
The present invention provides for a rescue method of recombinantly producing mumps virus. The rescued mumps virus possesses numerous uses, such as antibody generation, diagnostic, prophylactic and therapeutic applications, cell targeting, mutant virus preparation and immunogenic composition preparation. Furthermore, there are a number of advantages to using a recombinantly produced Jeryl Lynn strain of mumps for these applications. Some of these advantages include (1) an attenuated phenotype, (2) a substantial safety record based on the over 100 million dosages administered, (3) the ability to induce long-lasting immunity with a single dose and (4) a relatively low level of genome recombination.